1. Field of the Invention
The invention relates to the field of treatment of whey with a view to extracting valuable products.
2. Description of the Prior Art
Whey is a well-known by-product of the cheese-making industry. The composition of whey is approximately that of skim milk without its casein. In general, two main types of whey may be distinguished, sweet wheys, or cheese plant wheys, and acid wheys or casein plant wheys.
The addition of rennet to milk causes syneresis which results in a whey called rennet whey. If the renneting occurs at a pH of milk or at a slightly lower pH for example after slight maturation by lactic yeasts, but above a range of about 5.8 to 6, the whey is designated sweet whey. Moreover, the acidification of milk either by adding a mineral acid or by producing lactic acid (seeding milk with lactic ferments) at a pH near the isoelectric point of the caseins, causes the flocculation or coagulation thereof. After separation of the curd, an acid whey is obtained.
Whey is therefore defined with respect to the nature of the coagulation of the milk. In the cheese industry most of the wheys are in fact mixed wheys where one of the coagulation processes prevails over another. The sweet wheys come from the manufacture of cheeses called (cooked or uncooked) pressed curd (Emmental, Gruyere, Cheddar, Cantal, Saint-Paulin etc . . . ). Acid wheys are chiefly the by-products of the manufacture of fresh curds and casesin plants. Intermediate varieties are also found which are the by-products of the fabrication of most soft curds and marbled curds (blue cheese). Thus, the composition of wheys is subject to wide variations which mainly depend upon the original milk and the cheese-making procedures used.
Wheys have interesting constituents,, notably the nitrogenous fraction which essentially comprises soluble milk proteins which have an elevated biological value, greater than 90%. Reference can be made to the article by E. FORSUM and L. HAMBRAEUS, Nutritional and Biological Studies of Whey Products, J. of Dairy Sc., 60 (3), 370-377, 1977. Heretofore the three traditional destinations of wheys were spreading on fields, dumping in waterways and feeding animals. But the interest is now in new technologies involving, for example, ultrafiltration which permits separation, concentration and purification of the whey components selectively and modification of their physical-chemical properties while maintaining and even improving their nutritional quality. At the present time more and more research is directed to techniques of treating whey permitting the preparation of novel and varied products capable of finding uses as foodstuffs. Workers in the field have a particular interest in the proteins contained in the whey in order to obtain varied food products capable of satisfying specific requirements.
Numerous documents of the prior art illustrate treatments of whey by ultrafiltration. In the cheese-making field it has already been proposed to ultrafiltrate the whey to produce a "retentate" (which is concentrate held back by the ultrafiltration membrane) containing soluble proteins, the retentate then being reintroduced into the cheese in the process of fabrication. Yet there are whey ultrafiltration processes which permit differential or selective separation of various components. The process according to the present invention falls into the latter category.
An object of the invention is a process for obtaining from whey by ultrafiltration, a .alpha.-lactalbumin enriched product. Although in relative terms the proteins represent a small part of the solids content of the whey (less than 12%) they are the main attraction for increasing the value of this by-product. The proteinaceous fraction essentially comprising soluble milk proteins: .beta.-lactoglobulin, .alpha.-lactalbumin, serum albumin and immunoglobulins, is interesting by reason of its nutritional value and its fonctional properties.
Numerous works exist on the characterization of the soluble proteins in whey. As regards .beta.-lactoglobulin reference may be made to the work by G. Braunitzer, R. Chen, B. Shrank, A Stangl, Automatic Sequence Analysis of Protein (.beta.-lactoglobulin AB), Hoppe Seyier's Z. Physiol. Chem. 353 (5), 832-834, 1972. Briefly, it may be observed that the .beta.-lactoglobulin, by reason of the presence of a free SH group, has the tendency to polymerize and to exist in the form of a monomer-dimer-octamer equilibrium. The dimer from prevails in general in ultrafiltration conditions and at low temperature at pHs closer to the isolectric point (pH about 5 favors octamerization). At a pH greater than 7.5 the balance tips in favor of the monomer form. The conformation of the .beta.-lactoglobulin is relatively stable below pH 7. H. A. McKenzie, Milk Proteins, vols 1 and 2, Academic Press, New York, 1970, made a study of the forms and characteristics of .beta.-lactoglobulin. The effect of the pH on the conformation of this protein has been confirmed (see, for example, E. Mihalyi, Application of Proteolytic Enzyme to Protein Structure, 1972).
As for .alpha.-lactalbumin its sequence is generally known, see K. Brew, F. J. Castellino, T. C. Vanamam, R. L. Hill. The Complete Amino Acid Sequence of Bovine .alpha.-Lactalbumin, J. Biol. Chem. 245 (17), 4570-4582, 1973. Contrary to .beta.-lactoglobulin it does not have a free SH group. The work of D. N. Lee and R. L. Merson "Prefiltration of Cottage Cheese Whey to Reduce Fouling of Ultrafiltration Membranes", J. Food Sc. 41: 403-410, 1976, has demonstrated that the form of the .alpha.-lactalbumin molecules may vary in accordance with the pH. At acid pHs the molecules have the tendency to associate in dimer or trimer form. At pHs greater than 8 a polymerization of the protein is also found.
Like .beta.-lactoglobulin, serum albumin possess a free SH group and exists in monomer and dimer forms.
It is also know that the coagulation of milk is obtained by acidification or proteolysis of the K casein. This enzymatic hydrolysis with rennet frees from the whey a phosphoglycopeptide called caseinomacropeptide (CMP). The quantity of CMP in wheys varies according to the nature and the coagulation time, it may attain at the utmost 1 g/l in sweet whey.
Numerous other soluble proteins exist in milk and wheys. Their presence in smaller quantities than the preceeding makes them less important by their contribution ot the physical-chemistry of the protein concentrate of whey than their biological role.
The oldest technique for extracting proteins from whey consists of making them insoluble by a denaturing heat treatment at a pH close to their isoelectric point. This process, which is widely used was recently reviewed by B. P. Robinson, J. L. Short, K. R. Marshall, Traditional Lactalbumin Manufacture, Properties and Uses, N. Z. J. Dairy Sc. and Techn., 11 (2), 114-126, 1976. The drawback of this technique is obviously its denaturing aspect. Other laboratory processes have essentially tried to remedy this drawback. It has, for example, been proposed to carry out an adsorption of proteins in an ion exchanger (J. B. Ward, Separation Processes in the Biological Industry, Process Biochemistry, 11 (7), 1976. The ion exchange technique entails automation and continuous operation difficulties,, great investments (amounts of resin eluent, concentration of eluates). The obtention of purified products is not always an advantage.
Other processes call upon chemical extractions and do not give total satisfaction from the nutritional standpoint. Chromatographic ion exchange processes have also been suggested (see B. Mirabel, Nouveau procede d'Extraction des Proteines du Lactoserum, Ann. de la Nutrition et de l'Alimentation, 34 (2-3), 243-253, 1978. Filtration through a gel has also been used, but essentially in the laboratory. R ference may be had, for example, to the work of L. O. Lindquist and K. W. Williams, Aspects of Whey Processing by Gel Filtration, Dairy Industries, 38 (10), 459-464, 1973, and the work of E. Forsum, L. Hambraeus, and I. H. Soddiqt, Large Scale Fractionation of Whey Protein Concentrates J. of Dairy Sc., 57 (6), 659-664, 1974. The processing by gel filtration has a number of drawbacks. It requires, notably, a preconcentration of proteins which must not be denatured at the risk of altering the resolution between the different fractions. These fractions must then be concentrated and dried. The inferior mechanical properties of gels and the clogging capacity of whey concentrates by reason of the presence of lipoprotein complexes have made this procedure very limided.
Ultrafiltration through a membrane, given the progress made with regard to both apparatus and their performance, has become widely used in the dairy industry both for treating milk and whey (see, for example, the work of Mocquot et al, Preparation de Formage a Partir de "Prefromage liquide" obtenu par Ultrafiltration du Lait, Le Lait, 51 (508) 495-533, 1971.
During the filtering of whey through an ultrafiltration membrane, the water, soluble mineral salts and water-soluble vitamins pass through the membrane. The product which passes through the membrane is known as the ultrafiltrate or permeate. On the other hand the proteins and associated constituents (calcium, phosphorus), fat globules and lipophilic elements are held back or retained and are concentrated as the aqueous phase is eliminated. These constituents are known as the "retentate" or protein concentrate. The protein concentrates obtained by ultrafiltration may be defined either by the concentration factor or by the degree of purity of the proteins. The obtention of high purity concentrates necessitates the application of ultrafiltration followed or accompanied by diafiltration which consists in washing the protein concentrates. During diafiltration the liquid to be ultrafiltrated is brought into contact with the membrane at the same time as the solution, e.g., an aqueous solution. Numerous studies have been made on the composition of the whey ultrafiltrate. [See, for example, Hargrove et al. Production and Properties of Deproteinized Whey Powders, J. of Dairy Sc., 59 (1), 5-33, 1976, Hiddink et al Ultrafiltration of Condensed Whey, Zuivelzicht 68 (48-51), 1064-1066, 1126-1127, 1978; L. Kivieniemi, Microbial Growth During the Ultrafiltration of Sweet Whey and Skim Milk, Kemia-Kemi, 12, 791-795, 1974]. The composition of retentates or protein concentrates has also been determined. (See, for example B. S. Horton, R. L. Goldsmith and R. R. Zall, Membrane Processing of Cheese Whey Reaches Commercial Scale, Food Technol. 26 (30), 1972.
The actual conditions of ultrafiltration are such that it does not proceed according to ideal hypotheses. For instance, the ultrafiltration retentate has a relatively great residual content of fat as well as of mineral elements. Further, current ultrafiltration membranes have variable pore diameters. Their cut-off capacity is therefore not absolutely accurate and does not correspond to an ideal isoporous membrane. Furthermore the membrane is not inert. The temperature, pressure and pH may modify its physical properties and thereby the diameter of the pores and the hydration of the membrane may vary. Depending on the chemical nature of the membrane, ionic or hydrophobic interactions may develop between with the proteins and/or the minerals. In addition to these types of bonding one must add the possible physical capture of molecules in the pores of the membrane. All these phenomena modify the permeability of the membrane with respect to the ultrafilterable elements and water. Finally, it is necessary to underscore the importance of the formation on the ultrafiltration membrane of a polarization layer also known as a dynamic membrane which mainly comprises proteins. Without mastering this phenomenon any amelioration as regards selectivity of the membranes is largely compromised: see J. Murkes, Quelques Opinions sur les Applications Industrielles de la Technologie des Membranes, Journees Europeennes de la Filtration, Paris, Oct. 21, 1978. In other words the capacity of a membrane to satisfy a given function must not be appreciated ideally: the membrane must compulsorily be tested in operating conditions because it is impossible to disregard the polarization layer which forms in the course of ultrafiltration and modifies the transfer of solutes across the membrane, thereby lowering the performance of the apparatus, in particular, the permeation rate. Qualitative changes in the retentate and permeate may also be observed. This polarization layer serves as a second membrane and the total permeability of the membrane in the course of operation will depend on its thickness and also the nature of its components.
As regards the ultrafiltration of whey, reference may be had to the work by D. N. Lee and R. L. Merson, Prefiltration of Cottage Cheese Whey to Reduce Fouling of Ultrafiltration Membranes, J. of Food Sc. 41, 403-410, 1976. These authors determined a number of conditions which permit the thickness and density of the polarization layer to be minimized in order to improve the permeability of the membrane. They also noted the influence of pH which acts on the solubility of the proteins. It is therefore recommended to adjust the pH to take into account the origin of the whey. See also J. F. Hayes, J. A. Dunkerley, L. L. Muller and A. T. Griffin, Studies on Whey Processing by Ultrafiltration II Improving Permeation Rates by Preventing Fouling, The Australian J. of Dairy Tech. 37 (3), 132-140, 1974. The authors teach the preheating of whey, for example, to 80.degree. C. for 15 seconds, which will have the effect of improving the performance of the apparatus. Generally speaking it is also known to pretreat the wheys, i.e., heat treatment, notably by pasteurization to prevent bacterial contamination. Indeed it is known that whey contains 10 to 20% of the bacteria of milk manufactured. Pasteurization has therefore often been recommended to preserve the healthy quality of the whey.
The aforesaid articles by Lee and Merson (1976) and Hayes to which reference may be had if necessary, show that the conditions for ultrafiltrating wheys are of great practical importance.
Operations called diafiltration are also known, these operations consist of adding water to the retentate and simultaneously or subsequently eliminating an equivalent amount of permeate. The effect of diafiltration is to reduce the filterable elements from the retentate. It may be a batch process (dilutions followed by successive concentrations) or a continuous (water is added at the same rate as the permeate is eliminated). The diafiltration permits, in general, protein concentrates of enchanced purity to be obtained.
By way of example of references illustrating the prior art in the field of ultrafiltration the following may be cited: French patents Nos. 71.04.839 (printed publication No. 2.125.137) and 74.24.441 (printed publication No. 2.239.208), Chemical Abstracts, Vol. 81, No. 3, July 22, 1974, p. 218, Abstract No. 11969, referring to an article in Sci. Tecnol. Alimenti 1973, 3 (4) 209-215 by C. Pompei et al.
The French Pat. No. 71,04,839 relates to the delactosation of milk and proposes a process for producing a milk containing all the constituents of natural milks with the exception of lactose; such a product is useful for feeding diabetics. The disclosed process deals with the milk as a starting material and not whey, and it involves in addition at least one reverse osmosis step. Typically, the process according to French Pat. No. 71,04,839 consists in ultrafiltering milk, recovering the ultrafiltrate, subjecting the ultrafiltrate to reverse osmosis with a view to eliminating the lactose contained therein whereupon the effluent of the reverse osmosis is mixed with the ultrafiltration retentate to provide a reconstituted milk without lactose.
French Pat. No. 74,24,441 proposes to treat milk or whey by ultrafiltration by diluting the retentate with an aqueous solution in order to produce human milks in particular. After ultrafiltration of the whey a product which may be spray dried is obtained which after mixing with sodium caseinate results in an additive which is may be added to the milk to correct or modify the casein/soluble proteins ratio. Such a process therefore involves all the soluble proteins of whey. The article by C. Pompei et al. cited above also proposes treating whey by ultrafiltration to yield a protein concentrate which may be sprayed. Such a process enables all the whey proteins to be isolated but does not teach the fractionation thereof.
Observation of the prior art has shown that the retention of soluble whey proteins by the ultrafiltration membrane as classically used at the present time in industry is not complete. The retention also differs depending on the nature of the proteins.
Thus .alpha.-lactalbumin may pass freely through currently used membranes owing to its small molecular weight (14,000-15,000), its compact globular configuration and its inability to polymerize in a undenatured state by reason of the absence of free SH groups. Yet, in actual operating conditions of ultrafiltration a partial retention of .alpha.-lactalbumin is found which is surely due to the presence of the polarization layer of dynamic membrane on the actual membrane. As for .beta.-lactoglobulin which is also found to be partly retained, it passes, across the membrane in its monomer form. pH conditions therefore take on considerable importance.